fig1

Figure 1. Response of RAW 264.7 cells to LPS under glucose or galactose medium conditions. RAW 264.7 cells were plated in 6-well tissue culture plates (Corning, Corning, NY) and maintained in normal growth medium (NM) [DMEM (Gibco, ThermoFisher, Waltham, MA) containing 4.5 g/L glucose, 2 mM L-glutamine, sodium pyruvate, supplemented with 100 U/mL penicllin/ streptomycin (Sigma-Aldrich, Burlington, MA) and 10% fetal bovine serum (FBS #1 00-106, 0.25 endotoxin units/mL; Gemini Bio-Products, Sacramento, CA) and allowed to reach 85% confluence over 3-5 days. Cells were maintained at 37 °C, 5% CO2/5% O2, 90% humidity (Nu-5831 tri-gas incubator, Nuaire, Plymouth, MN). NM was changed to phenol-free NM medium or phenol-free DMEM medium containing 2 mM L-glutamine and 100 U/mL penicillin/streptomycin supplemented with (2) high glucose (25 mM; Glu), or 3] galactose (10 mM; Gla). Cells were maintained in the experimental medium for 3 days following which, under the same media conditions, cells were exposed to lipopolysaccharide (LPS; 1 μg/mL; Sigma) for up to 18 h and monitored using a live cell imaging system (IncuCyte, Sartorius) under normal incubator conditions. (A) Representative images (20x) of cell morphology under normal medium (NM), high glucose (Glu), or galactose (Gla). Scale bar = 25 microns. (B) Samples were collected at 3 h post-LPS and mRNA isolated using TRIzol® Reagent (Invitrogen, Carlsbad, CA), and 2.5 μL cDNA was used for qRT-PCR for Tnfa, II 1a, II 1b, and cyclophilin A using TaqMan^TM. Individual gene expression levels were normalized to cyclophilin A and presented as fold-change from vehicle controls in each medium condition. Data were analyzed by 2-way ANOVA followed by Dunnett’s test for independent group mean comparisons. Data represent mean +/- SEM (n = 3-4). *Significance level as compared to vehicle control set at P < 0.05